Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations

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Abstract

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.

Introduction

Ethambutol [dextro-2,2′-(ethylenediimino) di-1-butanol] (EMB) is a bactericidal, first-line drug for the treatment of tuberculosis (TB). EMB along with isoniazid, rifampin and pyrazinamide is used as an alternative to streptomycin, in the directly observed therapy short-course anti-TB regimen recommended by the World Health Organization and the Center for Disease Control and Prevention (CDC). Recent global data on drug resistance showed that resistance of Mycobacterium tuberculosis to streptomycin and EMB in newly diagnosed cases varied from 1 to 32% and 0 to 11%, respectively [1]. The primary resistance of M. tuberculosis strains to streptomycin and EMB in the United States was shown to be 6 and 2%, respectively [1]. The prevalence of primary and acquired resistance to EMB is generally much lower than that for streptomycin and is predominant among isolates from patients with multidrug-resistant-TB (MDR-TB). The early start of an effective therapy with at least two drugs to which the strain is susceptible is a key requirement for a positive treatment outcome in MDR-TB [2]. EMB is thus a valuable and alternative drug for streptomycin. Since resistance to EMB is generally associated with resistance to other antitubercular drugs, an early detection of embB resistance will not only abolish the risk of adverse reactions associated with EMB, particularly optic neuritis, it will also indicate the need to modify the therapy regimen.

Although the mechanism of action and the molecular genetic basis of resistance to EMB are complex and not fully defined, the drug interacts mainly with the membrane associated arabinosyltransferases as an arabinose analogue [3], [4]. The M. tuberculosis emb operon contains three contiguous genes namely embC, embA and embB encoding three homologous arabinosyltransferases [4]. Earlier studies involving EMB-resistant M. tuberculosis strains identified resistance-conferring amino acid substitutions in the embB gene in nearly 50 to 70% of the isolates [3], [4], [5]. The nucleotide substitutions most commonly occurred at codon position 306ATG-Met of embB gene. Five different mutations were found in this codon that altered its first or third base (ATG to GTG, CTG, ATA, ATC or ATT) resulting in the replacement of methionine by three different amino acids (Met to Val, Leu or Ile) [5]. Subsequent studies carried out on epidemiologically unrelated EMB-resistant M. tuberculosis strains not only showed the involvement of many more codons of the three arabinosyltransferases but also several other genes in EMB resistance [6]. However, this study also showed that the most frequent mutations occurred at embB codons 306, 406 and 497 and iniA codon 501 [6].

Many EMB-resistant M. tuberculosis strains can be rapidly identified by molecular methods that interrogate embB codons 306, 406 and 497 and iniA codon 501 [4], [5], [6]. However, elaborate and expensive methods such as DNA sequencing have been mostly used [4], [5], [6]. In this report, we describe simple and easily interpretable PCR-restriction fragment length polymorphism (RFLP) methods to detect, directly in BACTEC cultures, the most frequently found embB codons 306 and 497 and iniA codon 501 mutations in EMB-resistant M. tuberculosis strains.

Section snippets

Reference strains, clinical samples and DNA isolation

The M. tuberculosis H37Rv was used as the susceptible strain and well-characterized M. tuberculosis strains with specific substitutions at embB codons 306 and 497 and iniA codon 501, established by DNA sequencing of the DNA region around the respective codon positions, were used as reference strains. Twenty-five M. tuberculosis strains, isolated from TB patients, were obtained from the Chest Diseases Hospital, Kuwait. Isolation and identification of the clinical isolates was performed as

Results

The PCR performed on the DNA from the susceptible strain M. tuberculosis H37Rv (Fig. 1, Panel A, Lane Rv) and the mutant strains carrying ATG to GTG (Fig. 1, Panel A, Lane Mu1) or ATG to ATT (Fig. 1, Panel A, Lane Mu2) or ATG to CTG (Fig. 1, Panel A, Lane Mu3) mutation at embB codon 306 with primers EMB306A and EMB306B resulted in specific amplification of a 167 bp DNA fragment, as expected. The Nla III digested fragments of the amplified DNA from M. tuberculosis H37Rv resolved into four

Discussion

EMB was introduced as an effective anti-TB drug in the 1960s and is now used as an alternative drug for streptomycin. The worldwide prevalence of resistance to EMB is much lower than for streptomycin and is also predominant among patients with MDR-TB from some geographical locations/ethnic groupings [1]. Several studies have shown that majority of EMB-resistant M. tuberculosis strains contain mutations at embB codons 306, 406 and 497 and iniA codon 501 [4], [5], [6]. In particular, mutations at

Acknowledgements

We thank Z. Khan for technical assistance. This work was supported by Research Administration grant MI 06/02 and the College of Graduate Studies, Kuwait University.

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    Mutations at codon 378 (A → C, Glu-Ala) were common, however, it has been recognised as a phylogentically informative mutation [16] and only 2/10 (20.0%) strains with this mutation had phenotypic resistance to ethambutol. The role of mutations in codon 306 is uncertain, since it is commonly detected in ethambutol resistant and susceptible isolates [29–31]. Mutations in embB codons 306, 406 and 497 have all been associated with low level ethambutol resistance [32].

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